Thermodynamics of Protonation and of Copper(II) Binding in Aqueous

26 Thermodynamics of Protonation and of Copper(II) Binding in Aqueous Alginate Solutions Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 12, 2016 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch026

Alginic Acid from

Azotobacter

S. P A O L E T T I , A . C E S À R O , A . C I A N A ,

vinelandii F . D E L B E N , and G .

MANZINI

Institute of Chemistry, University of Trieste, Trieste, Italy V.

CRESCENZI

Institute of Physical Chemistry, University of Rome, Rome, Italy

The equilibrium properties in dilute aqueous solution of weakly ionized polysaccharides, e.g. carboxylated natural poly= saccharides, have not been so thoroughly investigated in comparison with other natural and synthetic polyelectrolytes. For instance, a detailed thermodynamic characterization of acid ionization and of counterion binding in terms of combined experimental potentiometric, calorimetric and volumetric data has not been achieved so far for the above types of polysaccharides. Such a description, however, is of obvious relevance for a better understanding of structure-conformation dependent solution properties for this important class of biopolymers. We wish to report here the results of a study on protonation equilibria and on copper(II) binding in aqueous alginate solution, performed using alginic acid from Azotobacter vinelandii (alginate S-35), whose solution properties have not as yet been studied in detail. Our data concern the changes in free energy, enthalpy (and entropy), as well as the volume changes associated with the uptake of protons or of Cu ions, respectively, by alginate S-35 macro= ions. 2+

Experimental Materials. Alginic acid (sample S-35) produced by Azotobacter vinelandii (1) was a kind gift of Dr.K.C.Symes, Tate and Lyle

0097-6156/81/0150-0379$05.00/0 © 1981 American Chemical Society In Solution Properties of Polysaccharides; Brant, David A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SOLUTION

380

PROPERTIES

OF

POLYSACCHARIDES

L i m i t e d , R e a d i n g , U.K. I t was p u r i f i e d b y d i s s o l u t i o n i n 0.Q5 M EDTA ( 0 . 2 % p o l y s a c c h a r i d e ) a n d c e n t r i f u g a t i o n f o r two h o u r s a t 20,000 g. The s u p e r n a t a n t was d i a l y s e d a g a i n s t 0.05 M EDTA a n d t h e n e x h a u s t i v e l y a g a i n s t p u r e w a t e r a t 5 ° C The i n t r i n s i c v i s c o s i t y o f t h e p u r i f i e d sample i n 0.2 M N a C l a t 25°C was 2.60 d l / g . F o r a l l measurements o n l y f r e s h l y p r e p a r e d s o l u t i o n s were u s e d . A l g i n i c a c i d c o n c e n t r a t i o n was d e t e r m i n e d b y p o t e n t i o m e t r i c t i t r a t i o n w i t h standard base u s i n g a l i q u o t s o f stock a l g i n a t e s o l u t i o n s , a f t e r c o n v e r s i o n t o f r e e a c i d by passage through anion exchange column ( H - f o r m ) , The t i t r e o f t h e s o l u t i o n s was a l s o r o u t i n e l y d e t e r m i n e d b y means o f N a i o n f l a m e p h o t o m e t r y . The r e s u l t s o f t h e two methods a g r e e d w i t h i n e x p e r i m e n t a l e r r o r ( c a . 2% o v e r a l l ) . C o p p e r ( I I ) p e r c h l o r a t e was p r e p a r e d b y r e a c t i o n o f t h e c a r b o n a t e w i t h warm aqueous HCIO4. The r e s u l t a n t s o l u t i o n was f i l t e r e d and c o o l e d : t h e m e t a l p e r c h l o r a t e p r e c i p i t a t e d was c o l l e c t e d a n d r e c r y s t a l l i z e d t w i c e f r o m w a t e r . The t i t r e o f t h e c o p p e r p e r c h l o r a t e s o l u t i o n s was d e t e r m i n e d u s i n g EDTA ( 2 ) . The e q u i l i b r i u m d i a l y s i s e x p e r i m e n t s were c a r r i e d o u t u s i n g c e l l u l o s e t u b i n g ( U n i o n C a r b i d e Co., C h i c a g o , USA). T r e a t m e n t o f membranes p r i o r t o u s e h a s b e e n p r e v i o u s l y r e p o r t e d ( 3 ) . A l l t h e r e a g e n t s u s e d were C . E r b a RP p r o d u c t s o f a n a l y t i c a l r e a g e n t g r a d e * HC1 s o l u t i o n s employed b o t h i n c a l o r i m e t r y and i n d i l a t o m e t r y were o b t a i n e d b y p r o p e r d i l u t i o n o f f r e s h l y p r e p a r e d s t a n d a r d Normex 0.1N HC1 s o l u t i o n s . The w a t e r employed h a d a s p e c i f i c c o n d u c t i v i t y o f c a . 10~6 XI ""Ι-αιΓ 1. I t was o b t a i n e d b y an a l l - g l a s s d e i o n i z a t o r bidistillator. +

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 12, 2016 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch026

+

M e t h o d s . D i l a t o m e t r i c measurements were p e r f o r m e d u s i n g C a r l s b e r g d i l a t o m e t e r s ( 4 ) . Both the procedure and the treatment o f d a t a have a l r e a d y been d e s c r i b e d 0 3 ) . The η-heptane employed as d i l a t o m e t r i c l i q u i d was a C . E r b a RP p r o d u c t : i t h a s been p u r i f i e d a s r e p o r t e d e l s e w h e r e ( 5 ) * The t h e r m o s t a t b a t h i n w h i c h t h e d i l a t o m e t e r s were s u s p e n d e d h a s a l r e a d y been d e s c r i b e d ( 6 ) ; c o n s t a n c y o f t e m p e r a t u r e was b e t t e r t h a n +0.001°C. The p o t e n t i o m e t r i c m e a s u r e m e n t s , u n d e r N2 f l u x , were p e r f o r m e d as p r e v i o u s l y d e s c r i b e d ( 7 ) . S i m i l a r l y , t h e e x e c u t i o n o f t h e e x p e r i m e n t s a n d t h e t r e a t m e n t o f t h e c a l o r i m e t r i c d a t a were t h e same a s a l r e a d y d e s c r i b e d i n d e t a i l e l s e w h e r e ( 7 ) . The e q u i l i b r i u m d i a l y s i s measurements were p e r f o r m e d b y i n t r o d u c i n g 2 m l o f 1.2x10"^ M s o d i u m a l g i n a t e ( i n 0*05 M NaCIO,) and 2 m l o f C u i C l O ^ ^ a t v a r i o u s c o n c e n t r a t i o n s ( i n 0.05 Μ N a C l O ^ i n t o t h e two compartments o f e a c h d i a l y s i s v e s s e l . I n a l l c a s e s d i a l y s i s e q u i l i b r i u m was r e a c h e d i n a b o u t 2 days ( a t 25°C) a n d t h e c o n c e n t r a t i o n o f c o p p e r ( I I ) i n b o t h compartments was d e t e r m i n e d . C o p p e r c o n c e n t r a t i o n s a s l o w as 10"^ M were d e t e r m i n e d a d d i n g a c o n s t a n t e x c e s s o f EDTA s o d i u m s a l t and m e a s u r i n g t h e a b s o r b a n c e a t 270 nm, u s i n g a s r e f e r e n c e a s o l u t i o n o f EDTA. a t t h e same c o n c e n t r a t i o n . I n t h e a b s e n c e o f o t h e r d i v a l e n t c a t i o n s t h e method p r o v e d r e p r o d u c i b l e w i t h i n +2% a n d t h e a b s o r b a n c e f o l l o w e d t h e

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

PAOLETTi E T A L .

Aqueous

I

Alginate

I

Solutions

I

381

I

ο

K

Pa 4.8


4.4

/

4.0 /

/

—

3.6

0

J

Ι

Ι

Ι

I

0.2

0.4

0.6

0.8

1.0

α

Figure 1. Apparent pK dependence on a for alginic acid S-35 in aqueous solu tion at 25°C, titrated with KO Η (Ο) and NaOH (Φ) respectively; polymer con centration, 10' equiv/L. a

3

0

0.2

0.4

0.6

0.8

^

10

Figure 2. Enthalpy change (kilocalories/'mole H") on proton dissociation of alginic acid S-35, partially neutralized with KOH (O) and NaOH (Φ) in water at 25°C; polymer concentration, 10~ equiv/L. 3


SOLUTION PROPERTIES OF

382

POLYSACCHARIDES

H

L a m b e r t - B e e r law up t o 5x10 M . The r e s u l t i n g a b s o r p t i o n c o e f f i c i e n t was 3.5x10^ WT-'-cm^ a t 270 nm ( t h e w a v e l e n g t h o f maximum a b s o r p t i o n b e i n g 260 nm). -

R e s u l t s and

Discussion

I) Protonation e q u i l i b r i a .

The

potentiometric K

titration +

d a t a a r e r e p o r t e d i n F i g u r e 1 a s a p l o t o f P ( = pH l o g — ) a g a i n s t t h e d e g r e e o f i o n i z a t i o n , (X . A l g i n a t e S-35 i n w a t e r i s a weak p o l y a c i d , c o m p a r a t i v e l y w e a k e r t h a n s t r u c t u r a l l y s i m i l a r p o l y s a c c h a r i d e s (8,9)· However, i t a p p e a r s f e a s i b l e t o e x t r a p o l a t e t h e e x p e r i m e n t a l c u r v e o f F i g u r e 1 s o t h a t t h e p K v a l u e common t o t h o s e a l g i n a t e s f r o m o t h e r s o u r c e s i s a t t a i n e d f o r 0( =0 ( 9 ) . The c a l o r i m e t r i c d a t a g i v e n i n F i g u r e 2 show t h a t t h e e n t h a l p y o f d i s s o c i a t i o n , Δ Η ^ £ , o f a l g i n i c a c i d S-35 i s a l w a y s n e g a t i v e ( e x c e p t f o r 0(£* 1) w i t h a d i s t i n c t minimum a t a r o u n d 0< = 0.6 o f a b o u t -2.2 k c a l / m o l H ( a t 25°C). C o n s i d e r i n g t o g e t h e r t h e p o t e n t i o m e t r i c and c a l o r i m e t r i c d a t a one c a n r e a d i l y e s t i m a t e t h a t


a

Q

8 8

+

i

H d l s s

the entropy of i o n i z a t i o n , ^ S (= ^ -2.3 R p K ) , i s a l w a y s n e g a t i v e w i t h a minimum v a l u e o f a b o u t -?8 e.u. a t CX = 0.6, w h i l e i t i s a b o u t -22 e.u. f o r 0< a p p r o a c h i n g u n i t y . I n o t h e r words, the Δ Η ^ and A S ^ £ values at near h a l f n e u t r a l i z a t i o n happen t o f a l l i n t h e i r r e s p e c t i v e n o r m a l r a n g e s f o u n d f o r o t h e r p o l y c a r b o x y l a t e s (10) as w e l l a s f o r monomeric c a r b o x y l a t e s ( 1 1 ) ; o n t h e c o n t r a r y t h e u p t u r n o f b o t h Δ Η and A S a t h i g h e r (X v a l u e s seems somewhat a n o m a l o u s . I n d e e d t h e p o t e n t i o m e t r i c d a t a a l s o seem t o d i s c l o s e a somewhat anomalous b e h a v i o r i n t h e same r a n g e o f h i g h OC v a l u e s . T h e s e o b s e r v a t i o n s might be r e l a t e d to the i o n i z a t i o n of c o n f i g u r a t i o n a l l y d i f f e r e n t -C00H g r o u p s w h i c h , p r o g r e s s i v e l y , c o u l d b r i n g a b o u t some c o n f o r m a t i o n a l p e r t u r b a t i o n along the a l g i n a t e c h a i n s . More r e m a i n s t o b e l e a r n e d w i t h t h e a i d o f d i f f e r e n t techniques on the c a u s e s , presumably c o n f o r m a t i o n a l ones, c o n t r i b u t i n g t o the o v e r a l l , p e c u l i a r t r e n d o f b o t h thermodynamic s t a t e f u n c t i o n s w i t h c h a n g i n g C< f r o m 0 t o u n i t y f o r o u r a l g i n a t e sample. On the o t h e r h a n d , t h e d i l a t o m e t r i c d a t a r e p o r t e d i n F i g u r e 3 do show t h a t t h e volume change o n d i s s o c i a t i o n o f a l g i n i c a c i d S-35 i s s i m p l y a l i n e a r f u n c t i o n o f CX . T h i s b e h a v i o r , r a t h e r uncommon f o r a p o l y e l e c t r o l y t e , d e m o n s t r a t e s t h a t d i s s o c i a t i o n o f i o n i z a b l e g r o u p s a l o n g a l g i n a t e c h a i n s t a k e s p l a c e w i t h o u t any i n t e r f e r e n c e among t h e h y d r a t i o n s h e a t h s o f s u c h g r o u p s . F r o m o u r d a t a one can i n f a c t c h a r a c t e r i z e a l g i n a t e S-35 w i t h j u s t one v a l u e f o r t h e d i f f e r e n t i a l volume o f d i s s o c i a t i o n , i . e . : -11.5 cnP p e r mole o f c a r b o x y l g r o u p s ( s l o p e o f t h e p l o t o f F i g u r e 3 ) . I n c o n c l u s i o n , i f a p H - i n d u c e d c o n f o r m a t i o n a l change i s a p o s s i b l e q u a l i t a t i v e e x p l a n a t i o n of the p o t e n t i o m e t r i c d a t a , such a change w o u l d h a v e no b e a r i n g o n t h e s o l v a t i o n o f a l g i n a t e S-35. I t has t o b e p o i n t e d o u t , m o r e o v e r , t h a t t h e i o n i z a t i o n b e h a v i o r d i s s

8

8

a

8 8



26.

PAOLETTi E T A L .

Aqueous

0

0.2

Alginate

0.4

383

Solutions

0.6

0.8

0£

1.0

+

Figure 3. Volume change in milliliters/equivalent on proton addition to the K (O) (ordinate right) and Na (Φ) (ordinate left) salt forms, respectively, of alginic acid S-35 in water at 25°C; polymer concentration, 2 X 10~ equiv/L in the former case and 2.5 X 10~ equiv/L in the latter one. The abscissae are to be read from the right to the left. +

3

3

(c l/monomol)

(ml/monomol)

a

Figure 4. Enthalpy changes (%) (left scale) in calories per mole of the polymer repeating unit, and volume changes (O) (right scale) in milliliters per mole of the polymer repeating unit, on the addition of Cu(ClO ) to the sodium salt form (a = 1.0) of alginic acid S-35 in 0.05M NaClO at 25°C. [Cu ]/[P] stands for the molar ratio of the total copper(II) to the polymer repeating unit. k

2+

h



384

SOLUTION

0.1

0.2

PROPERTIES

0.3 0.4 [

Cu

2

j

OF

POLYSACCHARIDES

b

[p] Figure 5. Scatchard plot from equilibrium dialysis measurements for the binding of Cu onto sodium alginate S-35 in 0.05M NaClO at 25°C. Polymer concentra tion, [P], was 1.2 χ 10~ equiv/L. [Cu +] and [Cu ] stand for bound and free copper molar concentrations, respectively. 2+

h

3

2

2+

b

f


PAOLETTi E T A L .

26.

Aqueous

Alginate

385

Solutions

o f t h i s p o l y c a r b o x y l a t e does n o t depend a t a l l on w h e t h e r c o u n t e r = i o n s a r e N a o r K i o n s (see F i g u r e s 1-3). 2+ I I ) B i n d i n g o f Cu i o n s . The r e s u l t s o f t h e c a l o r i m e t r i c experiments on the i n t e r a c t i o n of C u (added C u ( 0 1 0 4 ) 2 ) w i t h aqueous s o d i u m a l g i n a t e ( i n 0.05 M NaC104 s o l u t i o n ) a r e r e p o r t e d i n F i g u r e 4. The c a l o r i m e t r i c d a t a , e x p r e s s e d i n c a l o r i e s p e r s t o i c h i o m e = t r i e e q u i v a l e n t of p o l y e l e c t r o l y t e , A H g , as a f u n c t i o n of the s t o i c h i o m e t r i c [ ] C u 1 / N r a t i o (moles o f c o p p e r ( I I ) p e r e q u i v a l e n t o f a l g i n a t e i n s o l u t i o n ) , show t h a t ΔΗ-β i s a l w a y s p o s i t i v e and s t e a d i l y i n c r e a s e s w i t h i n c r e a s i n g added C u ion concentration. The d i l a t o m e t r i c r e s u l t s shown i n F i g u r e 4 c o l l e c t e d u n d e r experimental c o n d i t i o n s i d e n t i c a l to the c a l o r i m e t r i c ones, d e m o n s t r a t e t h a t t h e r i s a n e t volume i n c r e a s e upon b i n d i n g o f Cu2+ i o n s b y s o d i u m a l g i n a t e . F i n a l l y , a few e q u i l i b r i u m d i a l y s i s d a t a are r e p o r t e d i n F i g u r e 5 i n the u s u a l S c a t c h a r d - p l o t form. From the l a t t e r d a t a i t i s p o s s i b l e t o e s t i m a t e a v a l u e o f 4.10^ ( 1 / e q u i v ) f o r t h e a p p a r e n t e q u i l i b r i u m c o n s t a n t o f C u b i n d i n g , on t h e h y p o t h e s i s t h a t i t s i m p l y i n v o l v e s one f i x e d a l g i n a t e charge per C u i o n bound and no i n t e r d e p e n d e n c e between b i n d i n g s i t e s . C o n s e q u e n t l y , one a l s o e s t i m a t e s t h a t : Δ = -4.9 ( k c a l p e r mole o f b i n d i n g r e a c t i o n ) , and t h a t i n t h e c a l o r i m e t r i c and d i l a t o m e t r i c e x p e r i m e n t s a t , s a y , £ C u J / N = 0.05 more t h a n 90% o f the C u ions present i n s o l u t i o n are l i n k e d to a l g i n a t e chains. D e r i v i n g from the l i m i t i n g s l o p e s o f the p l o t s of F i g u r e 4 _ t h e d i f f e r e n t i a l e n t h a l p y ( Δ Hg) and t h e d i f f e r e n t i a l volume ( Δ ν ) o f _ b i n d i n g , r e s p e c t i v e l y , one t h e n f i n d s : Δ Η β = 2 k c a l , _ a n d Δν^ = 39 c n P , a l w a y s p e r mole o f c o p p e r bound. A l s o : A S g = 23 e.u. ( p e r mole o f c o m p l e x ) . 2+ I n c o n c l u s i o n , i t c l e a r l y a p p e a r s t h a t Cu ions are e x t e n s i v e l y bound b y a l g i n a t e S-35 and t h a t t h e p r o c e s s i s e n t i r e l y e n t r o p y d r i v e n . The l i n k b e t w e e n t h e changes i n Δ V and ΔS may b e r e a d i l y , a l t h o u g h q u a l i t a t i v e l y , e s t a b l i s h e d a s s u m i n g , i n a n a l o g y w i t h s i m i l a r e v i d e n c e f o r o t h e r p o l y c a r b o x y l a t e s 03), t h a t i n t e r a c t i o n between -C00" g r o u p s and C u ions loosens a r e l a t i v e l y l a r g e number o f w a t e r m o l e c u l e s f r o m t h e s o l v a t i o n s h e l l s of both i o n i c s p e c i e s , thus producing a net gain of _ m o l e c u l a r d e g r e e s o f f r e e d o m i n t h e s y s t e m . T h i s o c c u r s a t LCu2+J/ Ν r a t i o s n o t h i g h e r t h a n a b o u t 0.4, i n a s m u c h a s r e l a t i v e l y h i g h e r Cu i o n c o n c e n t r a t i o n s l e a d t o g e l f o r m a t i o n and h e n c e t o s y s t e m s i n t r a c t a b l e w i t h our e x p e r i m e n t a l approaches. I t w i l l be i n t e r e s t i n g t o make a c o m p a r a t i v e a n a l y s i s o f t h e t h e r m o d y n a m i c s o l u t i o n p r o p e r t i e s of a l g i n a t e samples o f d i f f e r e n t o r i g i n c o n t a i n i n g d i f f e r e n t p r o p o r t i o n s o f m a n n u r o n i c and g u l u r o n i c a c i d s as w e l l as o f t h e p a r e n t homopolymers. Work i s now u n d e r way a l o n g t h e s e l i n e s i n our l a b o r a t o r y . +

+

2 +


2 +

2 +

2 +

2 +

2+

2 +

β

B

B

2 +

2 +


386

SOLUTION PROPERTIES OF POLYSACCHARIDES

Acknowledgements. This work has been carried out with financial support of the I t a l i a n Consiglio Nazionale delle Ricerche, C.N.R. Literature cited 1. Sandford, P.A. and Laskin, Α., Eds, "Extracellular Microbial Polysaccharides"; ACS Symp. 45, American Chemical Society : Washington, D . C . , 1977; p.14. Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 12, 2016 | http://pubs.acs.org Publication Date: April 21, 1981 | doi: 10.1021/bk-1981-0150.ch026

2. Flaschka, H. Mikrochemie, 1952, 39, 38. 3. Crescenzi, V . ; Delben, F.; P a o l e t t i , S.; Skerjanc, J . J.Phys. Chem., 1974, 78, 607. 4. Linderstrøm -Lang, K. and Lanz, H. Comp.Rend,Trav.Lab.Carlsberg, Sez.Chim., 1938, 21, 315. 5. Katz, S. and F e r r i s , T.G. Biochemistry, 1966, 5, 3246. 6. Delben, F. La Chimica & l'Industria, in press. 7. Fenyo, J . C . ; Delben, F . ; P a o l e t t i , S.; Crescenzi, V. J.Phys. Chem., 1977, 81, 1900. 8. Mandel, M. J.Polymer Sci., A-2, 1970, 8, 1841. 9. Haug, A. Acta Chem.Scand., 1961, 15, 950. 10. Crescenzi, V . ; Delben, F.; Quadrifoglio, F . ; Dolar, D. J.Phys. Chem., 1973, 77, 539. 11. Delben F. and Crescenzi, V. J.Sol.Chem., 1978, 7, 597. R E C E I V E D August 26,

1980.


Thermodynamics of Protonation and of Copper(II) Binding in Aqueous

Recommend Documents