3 Investigation on Conformational Properties of Xanthan in Aqueous Solutions Downloaded by UNIV OF NORTH CAROLINA on September 25, 2014 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch003
M. MILAS and M . RINAUDO Centre de Recherches sur les Macromolécules Végétales, Laboratoire propre du C.N.R.S., associé à l'Université Scientifique et Médicale de Grenoble, 53 X - 38041 Grenoble Cedex, France
The ordered conformation of the Xanthan chain is generally interpreted as an helix which is characterized by a melting temperature T depending on the polymer concentration, ionic strength, nature of the counterions (1-3). It seems that until now, the dependence of T on the degree of neutralization (or pH) of the polyacid form and on the molecular weight has not been investigated. M
M
The dependence of conformational transition on the pH. In this part of the work, we report the results of potentiometry and optical rotation measurements. At different temperatures we have measured (Figure 1a) the dependence of the apparent pK on the degree of dissociation α . The pK is defined as : a
t
a
where at is the total degree of dissociation ; αt = α + + α with α + the degree of autodissociation of the carboxylic site during H
N
H
, Cp the concentration of xanthan expres
the titration (α + = H
sed in equivalent per liter) and α the degree of neutralization. Under acid form of the polymer, one gets : α with α the initial degree of ionization of the acidic groups. One observes that the curves change with increasing temperature. Above 40°C a shift in the position of the maximum can be observed. A similar behavior is found for the optical rotation as a function of α and the temperature (Figure 2a). Consideration of both figures leads us to the conclusion that there is a conformational change when α increases. This transition takes place for a given mel ting dissociation degree ( α ) which decreases when the temperature increases. ( ( α ) is taken as half transition at[α]300 - 75° decig. cm ). A plot of ( α ) as a function of the tempe rature in Figure 2b shows a transition around 40°C. This N
t
t
t
t
M
=
t
-1
Μ
2
t
M
0097-6156/81/0150-0025$05.00/0 © 1981 American Chemical Society In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
26
SOLUTION PROPERTIES O F POLYSACCHARIDES
temperature separates i n f a c t the two realms of d i f f e r e n t behav i o r o f Xanthan s o l u t i o n s as shown i n Figure l a . Behavior above 40°C. The behavior i s that o f a p o l y e l e c t r o l y t e with a charge-driven conformational change as monitored by o p t i c a l r o t a t i o n . The values o f [a] increase f o r the values o f a between 0.3 and 0.5 due t o the conformational t r a n s i t i o n (Figure 2a). The p K changes (Figure la) are i n agreement with the [of| curves and show a conformational t r a n s i t i o n with v a r i a t i o n of the charge p o t e n t i a l . In t h a t range of temperatures both the i n i t i a l degree of d i s s o c i a t i o n (Figure lb) and the corresponding pK vary only s l i g h t l y with temperature. In the presence o f an excess of s a l t (Figure 3a) the p K i s independent o f the degree of t i t r a t i o n and equal t o 3.3 ; a c o r r e c t i o n based on the DebyeHuckel theory f o r the i o n i c strength gives an i n t r i n s i c p K =3.14 for the c a r b o x y l i c groups. The i n i t i a l degree o f d i s s o c i a t i o n i s around 0.5 j u s t as with other c a r b o x y l i c polysaccharides (£) i n the range of polymer concentration i n v e s t i g a t e d . Above 40°C the behavior i s q u i t e normal and c o n t r o l l e d by the main h e l i x - c o i l t r a n s i t i o n of the p o l y s a c c h a r i d e . This i s not the case when the temperature i s lower ; thus when the o p t i c a l r o t a t i o n of the a c i d i c form o f Xanthan [ a ] i s i n v e s t i g a t e d , there i s a f i r s t small t r a n s i t i o n around 40°C and the value [CX]H i s i n agreement with a h e l i c a l s t r u c t u r e i r r e s p e c t i v e o f the presence or absence of e x t e r n a l s a l t (Figure 3b) ( [a] under c o i l form i s around -50° d e c i g . ~ l c m ) . t
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a
a
a
Q
H
2
Behavior below 40°C. The p K values are lower f o r low a and at the same time the i n i t i a l apparent degree o f d i s s o c i a t i o n i s high (Figure 1). In Figure l b , the i n i t i a l degree o f d i s s o c i a t i o n ag i s p l o t t e d as a f u n c t i o n o f the temperature. A transition i s confirmed around 30°C. In excess s a l t a t 10°C, the p K i s e x c e p t i o n a l l y low (pK = 2.8 and the corresponding i n t r i n s i c p K should be lower than 2.64 and the i n i t i a l degree o f d i s s o c i a t i o n ag i s very high i n absence of e x t e r n a l a c i d added. From o p t i c a l r o t a t i o n , i t i s c l e a r t h a t the secondary s t r u c ture i s much more s t a b l e a t temperatures below 40°C than above 40°C ; the degree o f d i s s o c i a t i o n necessary f o r melting a t 10°C i s 0.7 (Figure 2 ) . From t h i s s e t o f experimental r e s u l t s , i t i s suggested that hydrogen bonds e x i s t a t low temperature. Intramolecular bonds could s t a b i l i z e the secondary ordered s t r u c t u r e , which e x i s t s a t low temperature even when the apparent degree of d i s s o c i a t i o n reaches t o 0.7. Such hydrogen bonds are presumed to i n v o l v e the c a r b o x y l i c and hydroxyl groups i n the chain. This would i n c r e a s e s the l a b i l i t y o f the proton and correspond t o a decrease of the a
t
a
a
Q
In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
3.
MILAS A N D RINAUDO
Conformational
Properties
27
of Xanthan
Figure 1. (a) Apparent pK of the carboxylic groups as a function of the total degree of dissociation (a ) at different temperatures in water (C = 7.10* equiv/L); (b) initial degree of autodissociation of the poly acid a ° as a function of the temperature. a
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t
p
H
\ 10
30
50
70
t\
. . 70*c
Figure 2. (a) Specific optical rotation [a] as a function of the total degree of dissociation (a ) at different temperatures in water (C = 7.10 equiv/L); (b) the degree of dissociation (a )M (corresponding to the conformational change) as a function of the temperature. The data on Figure 2b have been obtained from Figure 2a.
. 60 . 50 • 40 . 30 * 20 . 10
300
t
4
v
t
K
r
,300
10
. NaCl .1 N * 2°
Wu
H
30
5 0 70
'PKa _3.5
70* ' Figure 3. (a) Apparent pK of the carboxylic groups in excess of external salt (NaCl 0.1N) at 10°C and 70°C (C = 7.10 equiv/L); (b) optical rotation [a] (acidic form) as a function of the temperature in water and in 0.1N NaCl. fC = 7.10' equiv/L). a
10V
/
v
4
300
H
4
p
In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
SOLUTION PROPERTIES OF
28
POLYSACCHARIDES
S t r u c t u r e of the s o l u t i o n and dependence on the molecular weight of Xanthan. The d i f f e r e n t molecular weights of Xanthan were obtained by p a r t i a l enzymic h y d r o l y s i s (5_) . I t i s shown i n Figure 4 that the melting temperature T decreases with decreasing molecular weight although the slopes of the curves T M = f d o g ut) which c o r r e l a t e with the heat of melting and with the charge parameter, are i d e n tical. This dependence i s probably due to a cooperative conformat i o n a l t r a n s i t i o n as suggested by Holzwarth (6_) the degree of coop e r a t i v i t y decreases with a decrease i n the molecular weight. As p r e v i o u s l y d e s c r i b e d , when the conformation i s ordered (as evidence by the o p t i c a l r o t a t i o n [a]) the molecule i s r i g i d and the s o l u t i o n becomes a n i s o t r o p i c ; t h i s b i r e f r i n g e n c e appears between crossed p o l a r i z e r s . In Table I, the c r i t i c a l concentrat i o n s ( i n the absence o f e x t e r n a l s a l t ) (C ) f o r the appearence of h e l i c a l conformation and (C**) f o r the appearence of b i r e fringence at 25°C are given as a f u n c t i o n of d i f f e r e n t molecular weights.
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M
TABLE I
\
C r i t i c a l concentrations at 25°C of ordered s t r u c t u r e formation as a f u n c t i o n of the molecular weights.
C* g/1
C** g/1 (birefringence) (b)
(a)
5
20 ± 3
10
5
10 ± 2
7
10
5
7 ± 1
8 ± 2
3.2
10
6
3 ± 0.5
3 ± 1
1.4
10
2
33 ± 3 22,5
± 2
(a) from the curve T = (log C ) when T = 25°C (b) from observation between crossed p o l a r i z e r s . M
p
M
The data i n Table I suggest that the ordered h e l i c a l conformation i s necessary f o r b i r e f r i n g e n c e to develop (which appears f o r a C** > c*) • For the higher molecular weights C** ^ C* clue to the i o n i c strength necessary to induce the h e l i c a l conformation at 25°C. The values of C** are a l i n e a r f u n c t i o n o f (M^)" (Figure 5); the o r g a n i z a t i o n o f the s o l u t i o n i s c o r r e l a t e d with the length of the molecule i n agreement with the model of F l o r y {7) . 1
In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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3.
MILAS A N D RINAUDO
Conformational
Properties
of
29
Xanthan
Figure 4. The temperature of conformational change for different molecular weight fractions of xanthan as a function of the total ionic strength (fi = $ C + C ), where $ is the activity coefficient of sodium xanthan in the absence of external salt and is equal to 0.65 (8) t
P
s
Figure 5. Critical concentration for the appearance of birefringence as a function of the inverse of the molecular weights
In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
30
SOLUTION
PROPERTIES
O F POLYSACCHARIDES
Experimental The commercial sample o f Xanthan has been p u r i f i e d and p r e c i p i t a t e d as the pure Na s a l t . I t s c h a r a c t e r i s t i c s were given i n a previous work (1_) . The a c i d i c form i s obtained by p e r c o l a t i o n through an i o n exchanger IR 120 H f o l l o w e d by p r o g r e s s i v e neutral i z a t i o n with NaOH. The c a r b o x y l i c content i s 1.58 x 10" equiv/g (Na form) i n good agreement with the c a l c u l a t e d value from the chemical s t r u c t u r e proposed by Lindberg (9_) . The pH i s measured using a TACUSSEL M i n i s i s 6000 a f t e r c a l i b r a t i o n with b u f f e r s o l u t i o n s pH ~ 7 and 4 a t each temperature. The anisotropy o f the Xanthan s o l u t i o n s i s observed between crossed p o l a r i z e r s . The o p t i c a l r o t a t i o n i s expressed as the spec i f i c rotation [ a ] determined a t a wavelength 300 nm i n a 10 cm quartz c e l l with a s p e c t r o p o l 1 from FICA.
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3
3 0 0
Literature 1 2 3 4 5 6 7 8 9
Cited
Milas, M., Rinaudo, M., Carbohydr. Res. (1979) 76 186-196 Holzwarth,G., Biochemistry (1976) 15 4333-4339. Morris, E.R., Rees, D.A., Young, G., Walkinshaw, M.D., Darke, A., J. Mol. Biol. (1977) 110 1-16. Rinaudo, M., Milas, M., J. polymer Sci Part C (1974) 12 2073-2081. Rinaudo, M., Milas, M., Int. J. of Biol. Macromol. (1980) 2 45-48. Holzwarth, G., Ogletree, J., Carbohydr. Res. (1979) 76 277-280. Flory, P.J., J. Polym. Sci. (1961) 49 105-128. Proc. Roy Soc. (1956) A 234 60-89. Rinaudo, M., Milas, M., Biopolymers (1978) 17 2663-2678. Jansson, P.E., Kenne, L., Lindberg, B. Carbohydr. Res. (1975) 45 275-282.
RECEIVED September 26, 1980.
In Solution Properties of Polysaccharides; Brant, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
