Investigations on Aqueous Solution Properties of -Carrageenans

25 Investigations on Aqueous Solution Properties of κ-Carrageenans M. RINAUDO

and C . R O C H A S

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch025

Centre de Recherches sur les M a c r o m o l é c u l e s V é g é t a l e s , Laboratoire propre du C.N.R.S., associé à l'Université Scientifique et M é d i c a l e de Grenoble, 53 X 38041 Grenoble Cedex, France

We established recently (1) that similarly to Xanthan (2) the melting temperatures for the helix-coil and sol-gel transitions in kappa carrageenan (Figure 1) are directly controlled by the total ionic concentration C . In Figure 2 the concentration dependence of the melting temperature T is determined from optical rotation and conductivities. On the heating and on the cooling curves, TM is given as a function of C with T

M

T

C

T

=

γ c + c P

S

C and C are the equivalent concentration of polyelectrolyte and external salt respectively and γis the mean activity coefficient of the counterions. From our previous work γ is calculated at half transition for equal proportions of coil and helical structures. From Figure 2, it is clear that the nature of counterions (K or Na ) has a strong influence on the stability of ordered conforma tions and on the formation of gel in kappa carrageenan. For each counterion there exists a critical ionic concentration C* above which hysteresis of optical rotation appears and a gel is formed. Above C* the melting temperatures corresponding to heating and cooling cycles become different. These experimental results con firm the data given by Rees and coworkers (3). P

S

+

+

1/ Helix-coil transition (CT < C*). In the first part of this work we investigate the influence of the macromolecular conforma tion (given by specific optical rotation [α] ) on the activity coefficient of Na and K (obtained by potentiometry). The temperature and the concentration dependence of the acti vity coefficient of the sodium form is very slight (Figure 3A). At infinite dilution γ is 0.72 ; the specific optical rotation [α]300 is constant corresponding to the coil conformation. We observe the same dependence for the potassium form at 35°C. But at 15°C a transition is observed both in γ and [α]300 (Figure 3A 300

+

+

Na

0097-6156/81/0150-0367$05.00/0 © 1981 American Chemical Society Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

368

PROPERTIES OF

POLYSACCHARIDES


SOLUTION

Figure 2. Variation of the melting temperature T with the logarithm of the free counterion concentration C (C = C + yC ): (O) cooling; (-\-) heating. OT

T

T

s

p

Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

25.

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κ-Carrageenans

and 3B). Over a given polymer concentration the h e l i x conforma t i o n becomes e s t a b l i s h e d . The values ( Y ~ Υ κ ) normalized for the h e l i x content are a l s o p l o t t e d on the same f i g u r e as Η 300 (Figure 3B) . The agreement i s good and proves that i t i s the conformational t r a n s i t i o n which induces the change of γ ; moreover the experimental values are i n agreement with Manning's theory (_4) . I f the charge parameter i s λ,the a c t i v i t y c o e f f i c i e n t i s given by the f o l l o w i n g r e l a t i o n s : +

+


N a

-I

λ . 1

In γ =

- In A

λ S 1

In γ = - y A

Assuming that λ(coil) i s c a l c u l a t e d from the chemical s t r u c t u r e (5_) and that λ (double h e l i x ) i s c a l c u l a t e d from X-ray data (6_) we get : calculated λ ( c o i l ) =0.68 -> γ . =0.71 coil calculated _ λ double h e l i x = 1.65 -> γ , . = 0.37 double h e l i x Λ

Λ

Ί

π

Λ

Ί

The experimental values are γ c o i l =0.72 and γ ordered form ^0.35. These values agree w i t h a d i m e r i z a t i o n o f ordered form (double h e l i x ) . When C < C* the temperature dependence o f the conformation i n v e s t i g a t e d by o p t i c a l r o t a t i o n i s p e r f e c t l y r e v e r s i b l e , without hysteresis. The h e l i x - c o i l t r a n s i t i o n o f K carrageenan can be a l s o ob served by c o n d u c t i v i t y measurements (Figure 4A). The h e l i x con t e n t c a l c u l a t e d from the r a t i o ρ o f the c o n d u c t i v i t y o f the p o l y e l e c t r o l y t e t o t h a t o f a simple reference e l e c t r o l y t e corresponds w e l l w i t h the values o f o p t i c a l r o t a t i o n (Figure 4B). In c o n c l u s i o n i t i s apparent- t h a t a c o n c e n t r a t i o n and tempe r a t u r e r e v e r s i b l e h e l i x - c o i l t r a n s i t i o n takes p l a c e i n s o l u t i o n s of kappa carrageenans as soon as the t o t a l i o n i c c o n c e n t r a t i o n Op becomes lower than a c r i t i c a l value C*. This c r i t i c a l value de pends on the nature o f the c o u n t e r i o n s . T

+

2/ S o l - g e l t r a n s i t i o n (Or > C*). For Op l a r g e r than about 7.10~3 eq/1 ( K form) the h y s t e r e s i s sets i n as shown i n F i g u r e 2. Figure 5A shows the h y s t e r e s i s i n s a l t - f r e e polymer s o l u t i o n i n o p t i c a l r o t a t i o n (Cp = 1.79 10" e q / l ; C = 0.96 10" eq/1 a t h a l f t r a n s i t i o n ) ; the curves can't be obtained i n t h e i r e n t i r e t y due t o the b i r e f r i n g e n c e . I n c o n t r a s t the e n t i r e curves o f con d u c t i v i t y are obtained (Figure 5B). These curves suggest a two step mechanism on h e a t i n g and a continuous process on c o o l i n g w i t h a m e l t i n g temperature lower than f o r h e a t i n g . The study o f the r a t i o o f the c o n d u c t i v i t i e s ρ between the polymer and a simple e l e c t r o l y t e used as a r e f e r e n c e g i v e s the same r e s u l t as the con d u c t i v i t y , bu i t i s more p r e c i s e . This i s due t o the temperature +

2

2

T


SOLUTION

PROPERTIES

OF

POLYSACCHARIDES


370


Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981. 30

K

Figure 3B. Concentration dependence of specific rotation [a] o and helix content (%) at 15°C and 35°C for potassium and sodium κ-carrageenan in the absence of external salt (O); helix con tent (%) at 15°C on potassium κ-carrageenan calculated from the values (y + — JN^) of Figure 3A (+).


to

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PROPERTIES

OF

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Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981. p

2

3

2

Figure 4B. Temperature dependence of specific rotation [a] oo and helix content (% for potas sium κ-carrageenan in the absence of external salt (Ο); helix content (%) calculated from the values of the ratio ρ of conductivities (-{-). C = 1.05 χ 10' equiv/L; KCl, 0.65 X 10' equiv/L is used as the reference.



374

SOLUTION

PROPERTIES

OF

Figure 5 A. Temperature dependence of specific rotation [a] oo κ-carrageenan in the absence of external salt C = 1.79 X 10

POLYSACCHARIDES

3

p

2

of potassium equiv/L.



Figure 5B. Temperature dependence of equivalent conductivity Λ (expressed in Ω" cm equiv' ) and ratio ρ of conductivities. C = 1.79 X 10~ equiv/L; KCl, 10~ equiv/L is used as the reference. 1

2

1

2

p

2


SOLUTION

376

PROPERTIES

OF

POLYSACCHARIDES

independence o f ρ ( i n c o n t r a s t t o the c o n d u c t i v i t y , the r a t i o o f two c o n d u c t i v i t i e s taken i n the same range i s about independent of the temperature). I n a d d i t i o n the study o f ρ demonstrates t h a t for low temperature the r a t e o f g e l formation i s s m a l l and t h a t the c o n d u c t i v i t y remains lower f o r the h e a t i n g than f o r t h e coo l i n g cycle. When the v i s c o s i t y and the polymer c o n c e n t r a t i o n are lower (C = 0 . 1 7 10-2 / ! . c = 0.9 1 0 " eq/1 ; C = 1 0 " eq/1) the b i r e f r i n g e n c e disappears and the g e l formation i s r e v e r s i b l e w i t h i n the time o f the experiment ; a l s o the temperature dependence o f 2


p

e q

s

2

T

S i m i l a r types o f curves are g i v e n f o r a " f r e s h " g e l and a g e l aged 15 hours a t 21°C on Figure 7. The r e l a t i v e p o s i t i o n and am p l i t u d e o f both steps are m o d i f i e d by the ageing. Our i n t e r p r e t a t i o n of experimental curves (Figures 5, 6, 7) i s as f o l l o w s : - When the g e l i s heated, f i r s t the i s o l a t e d h e l i c a l chains melt a t a temperature T ^ ; i n a second s t e p , the aggregates o f h e l i c a l segments melt a t a temperature T j ^ . The weight f r a c t i o n of i s o l a t e d chains decreases during the ageing. - When the g e l i s c o o l e d , only one m e l t i n g temperature i s obtained near T^/ w i t h an i n i t i a l increase o f [a] which i s abrupter than when the s o l u t i o n i s heated. T h i s temperature confirms the two-step mechanism o f the h e a t i n g curve, b u t when the s o l i s cooled, f i r s t the c o i l forms h e l i c a l segments and immediatly t h e r e a f t e r the h e l i c a l segments g e l . M

Conclusion In t h i s work, o p t i c a l r o t a t i o n i n d i l u t e s o l u t i o n s o f kappa carrageenans i s used to i n t e r p r e t the a c t i v i t y c o e f f i c i e n t o f po tassium counterions and t o demonstrate a conformational t r a n s i t i o n a s s o c i a t e d w i t h an i n c r e a s e o f the charge parameter corresponding to a d i m e r i z a t i o n . Above a c r i t i c a l i o n i c c o n c e n t r a t i o n a thermoreversible g e l e x i s t s and a h y s t e r e s i s o f o p t i c a l r o t a t i o n and c o n d u c t i v i t y i s present. When the r a t e o f h e a t i n g i s low a two step mechanism f o r the g e l m e l t i n g i s demonstrated by both methods. The r e l a t i v e importance o f these steps i s c o r r e l a t e d t o the ageing o f the g e l and thought t o be due t o the m e l t i n g o f i s o l a t e d h e l i c a l chains and aggregates o f chains r e s p e c t i v e l y . The m e l t i n g temperature determined from the h e a t i n g curve g i v e n i n Figure 2 are mean v a lues obtained f o r h i g h e s t h e a t i n g r a t e and are approximate. Accu r a t e values o f T computed from h e a t i n g curves depend on a v a r i e ty of experimental c o n d i t i o n s i n c l u d i n g the h e a t i n g r a t e , ageing of the g e l and o t h e r s . In our o p i n i o n the new concept (_3) of s o l - g e l t r a n s i t i o n given by Rees and coworkers i s not enough t o i n t e r p r e t the s p e c i f i c r o l e o f counterions i n the double h e l i x s t a b i l i z a t i o n and conse quently i n the g e l f o r m a t i o n . M



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κ-Carrageenans

10

20

30

T

(°C)

Figure 6. Temperature dependence of specific rotation [a] oo and ratio ρ of con ductivities for potassium κ-carrageenan in the presence of KCl. C = 0.179 X 10~ equiv/L, C = 0.910' equiv/L; KCl, 10' equiv/L is used as the reference. 3

2

p

2

2

s

20

3 0

Τ

(°C )

Figure 7. Temperature dependence of specific rotation [a] oo of potassium κ-car rageenan in the presence of KCl. C = 0.179 X 10 equiv/L; C = 0.910~ equiv/L. (A) cooling curve; (B) heating curve (the heating is done directly after A);(C) heating curve after aging 15 h at 21° C after the melting. 3

2

p

2

s


378

SOLUTION PROPERTIES OF POLYSACCHARIDES

Experimental Materials. Kappa carrageenan, a copolymer of Ο-β-D galactopyranosyl-4-sulfate (1 -> 4)-0-3,6-anhydro-a-D-galactopyranosyl (1 + 3) as confirmed by NMR spectroscopy (7_) (Figure 1) was sup plied by Sigma and used after purification. The aqueous solutions (10 g/1) were precipitated by ethanol in excess of salt (1M NaCl) and washed seven times with an ethanol water mixture (80/20 V / V ) . The sodium and potassium forms were obtained by exact neutraliza tion of the acidic form by NaOH or KOH after percolation through an ion exchanger IR 120 H+. The experimental value of the capa city of the sodium form was 2.44 10~ ± 0.02 eq/1 in good agree ment with 2.45 10~3 calculated from the molecular structure (equi valent weight 408). —

3


w

a

s

Techniques. The specific rotation [ct] 300 determined at 300 nm with a FICA Spectropol 1 spectropolarimeter in a 5 cm thermostated cell. Activities of potassium and sodium were deter mined with Tacussel ion-selective glass electrodes ; a saturated calomel electrode was used as the reference. All e.m.f. measure ments were made with a Tacussel Minisis 6000 pH-meter. A poten tial reading constant over a 20 min. period was regarded as a reliable e.m.f. Calibration curves were obtained using pure NaCl or KCl solutions before and after each set of measurements. The temperature control of all measurements was 0.01°C Conduc tivity measurements were performed with platinized electrodes Tacussel CM 01 G and with a conductivity bridge Tacussel CD 78. A peculiarity of this bridge is its ability to measure the ratio between the conductivity of a sample and of a reference. The temperature dependences of the specific rotation [of] 300 and of the conductivities were performed with a constant cooling or heating rate of 17°C/h. Literature Cited 1. 2. 3. 4. 5. 6. 7.

Rochas, C. and Rinaudo, Μ., Biopolymers (1980) in press. Milas, M. and Rinaudo, Μ., Carbohydr. Res. (1979) 76, 189-196 Morris, E.R., Rees, D.A. and Robinson, G., J. Mol. Biol. (1980) 138, 349-362. Manning, G.S., J . Chem. Phys. (1969) 51, 924-933 Snoeren, T.H.N. Thesis (1976), Ede, (The Netherlands) Bayley, S.T., Biochem. Biophys. Acta (1955) 17, 194-205. Rochas, C., Rinaudo, M. and Vincendon, Μ., Biopolymers (1980) in press.

RECEIVED September 8,

1980.


Investigations on Aqueous Solution Properties of -Carrageenans

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