Effect of Tetra-Alkyl Ammonium Bromide on the Rheological and Thermal Properties of Kappa-Carrageenan and Agarose Gels

Polymer Journal, Vol. 22, No. 11, pp 991-999 (1990)

Mineo WATASE*, Katsuyoshi NISHINARI,**·t Peter A. WILLIAMS,** and Glyn 0. PHILLIPS**

* Faculty of Liberal Arts, Shizuoka University, Ohya, Shizuoka 422, Japan ** The North East Wales Institute of Higher Education, Deeside, Clwyd, CH5 4BR, UK

(Received April 16, 1990)

ABSTRACT: Dynamic viscoelastic measurements and differential scanning calorimetry were carried out for kappa-carrageenan gels containing tetra-alkyl ammonium bromide in order to clarify the structure and functional properties of kappa-carrageenan. The dynamic Young's modulus E' of the gels showed a maximum as a function of concentration of tetra-alkyl ammonium bromide at around 0.2 mol I- 1 . At any particular temperature E;,.., decreased in the following

order: E;,..,(Me4 N-)>E;.• .(Et 4 N-)>E;..,(Pr4 N-)>E;,.• .(Bu 4 N-). The temperatures Tm of the endothermic peak accompanying the transition from gel to sol in the heating DSC curves shifted to higher temperatures with increasing salt concentration. The order of the melting tempera ture of these gels was the same as in E;,..,, however, the enthalpy of melting showed the reverse order. Cations with smaller dynamic hydration number noHN increase the elastic modulus and the melting temperature of kappa-carrageenan gels more than those with larger noHN· KEY WORDS Kappa-Carrageenan / Agarose /Gel/ Tetra-Alkyl Ammoni- um Bromide / Rheology / Differential Scanning Calorimetry / Dynamic hydration Number /

Kappa-carrageenan and agarose are seaweed polysaccharides is considered as follows: (i) polysaccharides, and their chemical structures single or double helices are formed from resemble each other: Kappa-carrageenan con random coils by lowering the temperature of sists of o-galactose 4 sulfate and 3,6-anhydro the solution, (ii) these helices aggregate to form o-galactose, while agarose consists of o an ordered structure which is called the galactose and 3,6-anhydro-L-galactose. How junction zone or the crystalline region, and (iii) ever, their gelling properties are quite different: as a result, the three dimensional network is the gelling ability of kappa-carrageenan is formed. Therefore, these gels consists of increased by the addition of a small amount junction regions connected by long flexible of alkali metal ions, alkali earth ions 1 - 6 and chains. The junction zones are considered to guanidine hydrochloride. 7 The addition of be formed by hydrogen bonds. Kappa-carra these chemical reagents in excess quantity geenan contains sulfate esters, while agarose decreases the gelling ability of kappa-carra does not. Therefore, the above mentioned ex geenan. The gelling ability of agarose is not perimental findings were understood as fol influenced by alkali metal ions or alkali earth lows: The cations may shield the electrosta ions, but decreased by guanidine hydro tic repulsion between sulfate esters in kappa chloride. The gelling mechanism of these carrageenan molecules, and since the electro-

t Permanent Address: National Food Research Institute, Tsukuba 305, Japan.

991 M. WATASE et a/. static repulsion inhibits the formation of helices et al. 11 observed the analogous transitions in of kappa-carrageenan molecules and their the presence of bromide and chloride co aggregation and tight binding, the introduction anions. The effect of alkali metal ions, 12 - 14•18 of cations promotes the aggregation of and poly-hydric alcohols15 on the gel forming kappa-carrageenan molecules, and this in ability ofkappa-carrageenan and agarose have creases the gelling ability. Since agarose has no been studied by rheological measurements and sulfate esters, the cations have less influence on differential scanning calorimetry (DSC). the gelling ability. This interpretation was In the present work, the effects of tetra-alkyl confirmed by the effect of guanidine hydro ammonium salts on the rheological and chloride (GuHCI) and urea on rheological and thermal properties of Kappa-carrageenan and thermal properties of these gels. 7 Both of these agarose gels were examined in order to compare chemical reagents are hydrogen bond breakers. with the effects of other chemical reagents on However, GuHCI is an electrolyte, and so the these properties. The gelling ability of kappa guanidinium ions shield the electrostatic carrageenan will be influenced by two factors: repulsion between sulfate esters in kappa (i) tetra-alkyl ammonium salts will play a role carrageenan as alkali metal ions or alkali earth as the electrolyte and interact with kappa metal ions do. Therefore, the addition of carrageenan molecules, and (ii) they will change GuHCl below a certain concentration increases the structure of water as a solvent and therefore the gelling ability of kappa-carrageenan, but it will also affect the structure of the gels. decreases the gelling ability of agarose. Since urea is not an electrolyte, it only decreases the EXPERIMENTAL gelling ability of both polysaccharides. Tetra-alkyl ammonium salts have attracted Materials much attention from many investigators Kappa-carrageenan was extracted from because of their importance in the study of Chondrus crispus as used in the previous hydrophobic interaction and the iceberg report. 7 The intrinsic viscosity in 0.01 N formation. 8 NaSCN solution at 35°Cwas 14.0 (100ml g- 1). Grasdalen and Smidsrod2 •9 reported that the The potassium content in the specimen mea conformational transition of kappa-carrageen sured by atomic absorption spectrophotom an molecules detected by the change of specific etry was 3.24%. Agarose was extracted from optical rotation occurred by lowering the Gelidium amansii as used in the previous temperature in tetramethyl iodide but not in report. 7 It was pretreated with 3% NaOH, and tetramethyl chloride. This result together with then washed in flowing water. The intrinsic the invariance of molecular weights of viscosity in 0.01 N NaSCN at 35°C was 3.3 tetramethyl-ammonium kappa-carrageenate in (100 ml g- 1 ). The preparation methods used N(CH3) 4 salts led them to the adoption of a in this study were the same as reported pre single-stranded carrageenan-iodide complex viously_ 7, 12 - 1 s

rather than the double-helix structure that had Extra pure reagent grade (CH3) 4 NBr, 1 previously been proposed by Morris and Rees. (C2 H 5) 4 NBr, (C 3 H 7) 4 NBr and (C4 H 9 ) 4 NBr Norton et al. 11 also observed the order (Wako Pure Chemical Co., Ltd.) were used disorder transition by optical rotation mea without further purification. surement for tetramethyl ammonium kappa

carrageenate in the presence of (Me)4NC1, Measurements (Me)4 NBr and (Me)4 NI. Although Grasdalen The dynamic Young's modulus E' at 2.5 Hz and Smidsrod2•9 reported that this order was measured at various temperatures using a disorder transition is unique to iodide, Norton Rheolograph Gel CV-100 from Toyo Seiki

992 Polym. J., Vol. 22, No. II, 1990 Carrageenan and Agarose Gels with Tetra-Alkyl Ammonium Bromide

Seisakusho Ltd. The temperature was con experimental methods were described pre trolled by a silicone oil bath, and the gel sample viously .12 -15 was kept at each measurement temperature for 20 min. Differential scanning calorimetry RESULTS AND DISCUSSION (DSC) was carried out with a highly sensitive DSC SSC580 from Seiko Electronics Ltd. The Figures 1 and 2 show the dynamic Young's heating rate was fixed as 2°C min - i throughout modulus E' of2% w/w kappa-carrageenan gels the present work. The details of these as a function of concentration of tetra-alkyl

15------~

~E 10 'l'E z z 10

...0 ''o

X X w w

0 0 0,5

M~NBr cone. (mol/1) Et4 NBr cone. (mol /I) Figure I. The dynamic Young's modulus E' of 2% w/w kappa-carrageenan gels with and without tetra-alkyl ammonium bromide as a function of concentration of Me4 NBr (A) and Et4 NBr (B) at various temperatures: a, 15°C; b, 25°C; c, 35°C; d, 45°C.

8 (D)

"''E "IE z z 5 "o "o 4 X >< UJ w

0 0.5 I 0 0.2 0~ Pr4 NBr cone. (mol/1) 81J4N8r cone. (mol/ ll Figure 2. The dynamic Young's modulus E' of 2° w/w Kappa-carrageenan gels with and without tetra-alkyl ammonium bromide as a function of concentration of Pr4 NBr (C) and n-Bu4 NBr (D) at various temperatures: a, 15°C; b, 25°C; c, 35°C; d, 45°C.

Polym. J., Vol. 22, No. 11, 1990 993 M. WATASE et a/.

4~------, 4 ~------,

3 3 ~E "''E z z ., "'o 2 0 2

X X ' w 'w

o o.5 r 1.5 0 0.7 1.4 Me.iNBr cone. (mol/1) Et4 N Br cone. ( mol /I) Figure 3. The dynamic Young's modulus E' of 3% w/w kappa-carrageenan gels with and without tetra-alkyl ammonium bromide as a function of concentration of Me4 NBr (A) and Et4 NBr (B) at various temperatures: a, 15°C; b, 25°C; c, 35°C; d, 45°C, e, 55°C.

2 (D)

"''E 2 "''E z z

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>< X I w ' w

0 0 0.4 0.8 B1.4NBr cone. (mol/1 ) Pr4 NBr cone. (mol/1 J

Figure 4. The dynamic Young's modulus E' of 3% w/w kappa-carrageenan gels with and without tetra-alkyl ammonium bromide as a function of concentration of Pr4 NBr (C) and n-Bu4 NBR (D), at various temperature: a, 15°C; b, 25°C; c, 35°C; d, 45°C; e, 55°C; f, 65°C. ammonium bromide at various temperatures. slightly to higher concentrations with increas E' increased sharply up to 0.l--0.2mol 1- 1 ing temperature. concentration of tetra-alkyl ammonium bro Figures 3 and 4 shows the dynamic Young's mide and then decreased as the concentration modulus E' of 3 % w/w kappa-carrageenan gels increased further. The peak height of E' at the as a function of increasing concentration of same temperature was in the following order: tetra-alkyl ammonium bromide at various E;,,axCMe4N-) > E;,,ax(Et4N-) > E;,,axCPr4N-) > temperatures. General tendencies were the same E;,,ax

994 Polym. J., Vol. 22, No. 11, 1990 Carrageenan and Agarose Gels with Tetra-Alkyl Ammonium Bromide

3~------~------~---~

15 C\I 'E 25 z 35

"b 55 45

w I

65 65 (0) ( b) ( C)

0 0 0 0.6 cane. (mol/1) Figure 5. The dynamic Young's modulus E' of 2% w/w agarose gels with and without tetra-alklyl

ammonium bromide as a function of concentration of Me4 NBr (a), Et4 NBr (b), and n-Bu4 NBr (c). Figures beside each curve represent the temperature in °C. ammonium bromide at which E' becomes without tetra-alkyl ammonium bromide. An maximum shifted remarkably to higher con endothermic peak accompanying the transition centrations with increasing temperature in gels from gel to sol was observed in all cases. The containing Pr4 NBr (Figure 4 (C)) and Bu4 NBr peak temperature Tm shifted to higher (Figure 4 (D)). temperatures with increasing concentration of Figure 5 shows the dynamic Young's tetra-alkyl ammonium bromide. Norton et modulus E' of 2% w/w agarose gels containing at: 11 found that the order-disorder transition tetra-alkyl ammonium bromide as a function temperature of kappa-carrageenan gels shifted of the concentration of tetra-alkyl ammoni to higher temperatures with increasing tetra um bromide. The change of E' induced by methyl ammonium bromide by optical rotation tetra-alkyl ammonium bromide was smaller in measurements, which corresponds well to our comparison to kappa-carrageenan gels. The results shown in Figure 6(A). This shift was

values of E' for agarose gels scarcely changed most marked in gels containing Me4 NBr, and by the introduction of alkali metal salts12•13 then in the order Et4 NBr, Pr4 NBr, Bu4 NBr, because agarose does not contain any groups i.e., this shift became less marked in the order which are influenced by salts. However, the of the size of cations. It seems that too bulky change of E' for agarose gels induced by cations are not as effective in promoting the tetra-alkyl ammonium bromide is not neglig formation of junction zones. ible. E' of 2% w/w agarose gels as a function The DSC endothermic peak became sharper of the concentration showed a minimum up to a certain concentration of tetra-alkyl around 0.2----0.5 mo11- 1 concentration of ammonium bromide and then became broader tetra-alkyl ammonium bromide and this was beyond that concentration with increasing followed by a maximum in gels containing concentration of the salt. This corresponds

Me4 NBr and Et4 NBr. well to the rheological finding that E' increas Figures 6 and 7 show DSC heating curves ed up to a certain concentration of tetra of 2% w/w kappa-carrageenan gels with and alkyl ammonium bromide and then began to

Polym. J., Vol. 22, No. 11, 1990 995 M. WATASE et al.

~0.1(8) ~·'

,,0 UJ"

Jo.05mJ/s

40 60 30 50 90 Temperature ( °C )

Figure 6. Heating DSC curves of 2% w/w kappa-carrageenan gels with and without tetra-alkyl ammonium bromide. Figures beside each curve represent the concentration of Me4 NBr (A) and Et4 NBr (B) in mol 1- 1 •

o (D) ~·

..,0 0,2 UJ"

10,05mJ/s Jo.o5mJ/s

30 50 70 40 80 Temperature ( "C)

Figure 7. Heating DSC curves of 2% w/w kappa-carrageenan gels with and without tetra-alkyl ammonium bromide. Figures beside each curve represent the concentration of Pr4 NBr (C) and n-Bu4 NBr (D) in mo! I- 1• decrease with increasing concentration of dine hydrochloride. The appearance of gels the salt. This tendency has also been observ changes from solid-like or rubber-like to ed in kappa-carrageenan gels containing alkali liquid-like beyond a certain concentration of metal salt, alkali earth metal salt, and guani- the added chemical reagents. Since the DSC

996 Polym. J., Vol. 22, No. 11, 1990 Carrageenan and Agarose Gels with Tetra-Alkyl Ammonium Bromide endothermic peak is equivalent to the max imum of heat capacity, the gel-sol transition was explained recently by a zipper model approach. 16 The approach is based on a model dealing with the melting of thermoreversible gels whose junction zones mainly consist of crystalline regions resulting of association of rigid ordered molecular structure such as helices or extended rod-like molecules. A zipper is here a dimer of macromolecular chains formed by parallel links. According to this treatment, the DSC endothermic peak is determined mainly by the bonding energy E Figure 8. The gelation mechanism of kappa-carrageen which makes aggregate the ordered molecules an. Filled circles represent the tetra-alkyl ammonium ions. (a), random coil: (b), junction zone; (c), junction zone. (b) such as double helices or extended rigid corresponds to a sharp DSC endothermic peak, while (c) molecules, by the rotational freedom G oflinks corresponds to a broad DSC endothermic peak. around the aggregate, and by the number of bonds N. When the experimental DSC 2....------~ endotherm is sharp as in the case of kappa-carrageenan without any chemical re • agent, it is well reproduced by one set of E, G, 0.5 oe

and N. However, introduction of tetra-alkyl 0.3 ammonium bromide promotes the aggregation 0.2 of helical kappa-carrageenan molecules, and so 0 j the gel tends to a more ordered structure. 0.1 However, excessive salt immobilises free

water which is necessary for junction zone 0.05 formation, or hinders the aggregation of helices although the junction zone itself seems to become more heat-resistant. At the same time, 0.01..,...,,...___,...... _ _ _.__ _ __._ _ __,_ _ ___. __,____, the excessive salt seems to induce the 2.86 2.90 2.94 2.98 3.02 3.06 3.10 3. 14 distribution of E, G, and N. Therefore, the 103 · Tm" 1 / K DSC endothermic peak becomes broader in the presence of excessive salt. This situation may Figure 9. The relation· between the inverse of melting temperature Tm of 2% w/w kappa-carrageenan gels be represented in Figure 8. containing tetra-alkyl ammonium bromide and the Figure 9 shows the plot of log C against I/Tm logarithm of the concentration of tetra-alkyl ammonium for carrageenan gels, where C is the concentra bromide: O, Me4 NBr; e, Et4 NBr; t:,, n-Pr4 NBr; •• tion of the salt. According to Manning's n-Bu4 NBr. theory, 17 this plot will become a straight line at lower concentrations, and the slope of the '1H(Me4 N-), where '1H(Bu4 N-) represents the straight line obtained from this plot is enthalpy of metling of gels containing Bu4 NBr. proportional to the enthalpy '1H of melting of Gels formed in the presence of Bu4 NBr, the gels. As is seen from Figure 9, the plot therefore, have the lowest value for E;,.ax and deviates at higher concentrations, and the Tm' but the highest enthalpy of melting. This magnitude of '1H was in the following order: behaviour is contrary to that observed 18 19 '1H(Bu4 N-) > JH(Pr 4 N-) > '1 H(Et4 N-) > generally for polymer gels • and further

Polym. J., Vol. 22, No. 11, 1990 997 M. WATASE et a/.

Temperature ('t)

Figure 10. Heating DSC curves of2% w/w agarose gels with and without tetra-alkyl ammonium bromide. Figures beside each curve represent the concentration of Me4 NBr (A), Et4 NBr (B), and n-Bu4 NBr (C) in mo11- 1.

work is required in order to fully explain the 40 experimental results. NH/ Figure 10 shows the heating DSC curves of 30 2% w/w agarose gels with and without (J CII tetra-alkyl ammonium 0 0 bromide. The small ex (Et)4 W :a 20 (Me) N+ othermic peaks which appeared at about 30°C 4 0 (Pr)4 N+ were supposed to be induced by molecular ,::, rearrangement into a more ordered structure 10 (Bu)4 N+ during heating, which accompanies syneresis. These exothermic peaks appear only in the first 0 0 10 20 heating run, and disappear in the reheating 30 40 50 noHN DSC curves. The endothermic peak accompa nying the transition from gel to sol shifted Figure 11. Plot of dTm/d log C (C, concentration of slightly to higher temperatures in the case of tetra-alkyl ammonium bromide) for 2% w/w kappa carrageenan gels containing tetra-alkyl ammonium bro Me4 NBr and Et4 NBr, while it shifted to slightly mide as a function of the dynamic hydration number noHN lower temperatures in the case of Bu4 NBr. of cations. Figure 11 shows the plot of the value dTm/d log C taken from Figure 9 against the ability of kappa-carrageenan molecules. 1 - 6 If dynamic hydration number nottN, where noHN the electrostatic repulsion of sulfate groups in is defined as nh(r//rc°-1). 20 - 22 Here, nh is the kappa-carrageenan molecules is suppressed by coordination number, 'ch is the rotational cations, the formation of helices and their correlation time of water molecules around the aggregation is promoted, and hence the gelling ammonium salts, and 'c O is the rotational ability is increased. It was found that K + and correlation time of pure water. The dynamic Cs+ are more effective is increasing the elastic hydration number of the ammonium ion is modulus and shift the melting temperature Tm estimated as -0.55 by Uedaira.23 The melting to higher temperatures for kappa-carrageenan temperature decreased with increasing the gels than Li+ and Na+. 3 ' 12•13 The reason was dynamic hydration number of cations. It is well attributed to the fact that structure breaking known that cations such as alkali metal ions ions such as K + and Cs+ shield the electrostatic and alkali earth metal ions enhance the gelling repulsion of sulfate groups more effectively

998 Polym. J., Vol. 22, No. 11, 1990 Carrageenan and Agarose Gels with Tetra-Alkyl Ammonium Bromide than structure making ions such as Li+ and of Polysaccharides," D. A. Brant, Ed., Amer. Chem. Na+ because electrostatic shielding of structure Soc. Symp. Series 150, Washington D. C. American Chemical Society, 1981, p. 367. making ions is weakened by intervening water 6. D. Day, G. 0. Phillips, and P. A. Williams, Food molecules. The concept of dynamic hydration Hydrocolloids, 2, 19 (1988). number noHN is more quantitative than that of 7. M. Watase and K. Nishinari, Food Hydrocol/oids, 1, structure making or breaking ions; larger noHN 25 (1986). 8. W. Y. Wen, in "Water and Aqueous Solution," R. indicates structure making, while smaller noHN A. Horne, Ed., Wiley, New York, 1972, Chapter 15. indicates structure breaking. Therefore, the 9. H. Grasdalen and 0. Smidsrod, Macromolecules, 14, relation between noHN of tetra-alkyl ammoni 1842 (1981). um ions and d T ml d log C shown in Figure 11 10. D. A. Rees, E. R. Morris, D. Thom, and J. K. can be understood just as in the case where Madden, in "The Polysaccharides," for G. 0. Aspinall, Ed., Academic Press, New York, 1982, alkali metal ions are added to kappa Chapter 5. carrageenan solution and to increase the gelling I I. I. T. Norton, E. R. Morris, and D. A. Rees, Carbohydr. Res., 194, 89 (1984). ability. 3 •12•13 Cations with smaller dynamic 12. M. Watase and K. Nishinari, Rheol. Acta, 21, 318 noHN hydration number increase the elastic (1982). modulus and the melting temperature of 13. M. Watase and K. Nishinari, Colloid Polym. Sci., kappa-carrageenan gels than those with larger 260, 971 (1982). 14. M. Watase and K. Nishinari, Polymer J., 18, 1017 noHN· (1986). 15. K. Nishinari and M. Watase, Agric. Biol. Chem., 51, Acknowledgements. Authors thank Prof. 3231 (1987). H. Uedaira for valuable discussions on dy 16. K. Nishinari, S. Koide, P.A. Williams, and G. 0. namic hydration number. Phillips, J. Phys. (France), 51, 1759 (1990). 17. G. S. Manning, Biopolymers, 11 937 (1972). 18. M. Watase and K. Nishinari, Makromol. Chem., 188, REFERENCES 2213 (1987). 19. H. M. Tan, A. Moet, A. Hiltner, and E. Baer, I. E. R. Morris, D. A. Rees, and G. J. Robinson, Mo/. Macromolecules, 16, 28 (1983). Biol., 138, 349 (1980). 20. H. Uedaira, M. Ikura, and H. Uedaira, Bull. Chem. 2. 0. Smidsrod and H. Grasdalen, Hydrobiologia, Soc. Jpn., 62, I (1989). 116/117, 19 (1984). 21. M. Ishimura and H. Uedaira, Bull. Chem. Soc. Jpn., 3. M. Watase and K. Nishinari, J. Texture Stud., 12, 63, I (1990). 427 (1981). 22. H. Uedaira, "Hydrogels and Water," in "The Science 4. V. J. Morris and G. R. Chilvers, Carbohydr. Polym., of Food Hydrocolloids," K. Nishinari and T. Yano, 3, 129 (1983). Ed., Asakura Shoten, Tokyo, (1990). 5. R. Rinaudo and C. Rochas, in "Solution Properties 23. H. Uedaira, private communication (Sapporo).

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