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Determination of the Molar Mass Distribution of Lignins by Gel Permeation Chromatography Kaj Forss, Raimo Kokkonen, and Pehr-Erik Sågfors The Finnish Pulp and Paper Research Institute, P.O. Box 136, 00101 Helsinki, Finland

This paper describes the fractionation of lignin sulfonates on elution through Sephadex G-50, G-75 and Sephacryl S-300 using water or a 0.5M sodium chloride solution buffered to pH 8 as the eluent. Fractionation of kraft lignin on elution through Sephadex G-50 with 0.5M sodium hydroxide as well as the influence of the sodium hydroxide concentration is also described. By comparing the retention volumes of proteins and lignin sulfonate fractions with known molar masses, it is shown that several commercially available proteins can be used for calibration of the columns. It is shown that on elution through Sephadex G-25 with 0.5M sodium hydroxide the retention volume of monomeric compounds is influenced more by their functional groups than by their molecular size. A method is needed for the determination of the molar mass and molar mass distributions of lignins. The method should give reproducible results when used in different laboratories. The procedure should not be complicated, and the calibration components must be readily available and reasonably priced. The present paper describes the fractionation of lignin sulfonates and kraft lignin by gel permeation chromatography (GPC) and the method developed and used for several years at the Finnish Pulp and Paper Research Institute. Sulfonated Lignins Elution of Lignin Sulfonates with Water. As can be seen from Figures 1, 2, 3, and 4, the fractionation of lignin sulfonates on elution with water through Sephadex G-50 and G-75 takes place in such a way that the logarithms of 0097-6156/89A)397-0124$06.00/0 © 1989 American Chemical Society

ι

1.5

2.0

2.5

3.0 V / V

0

E F F L U E N T VOLUME

V.ml

Figure 1. Fractionation of lignin sulfonates in spent sulfite liquor on Sephadex G-50. Eluent: water. (Reprinted with permission from réf. 1. Copyright 1969 Wiley.)

1.0

1 I ι ι I ι ι I » I ι ι ι ι I ι ι ι I I

A280nm

1.5

2.0

2.5 V / V

0

EFFLUENT VOLUME V.ml ι ι ι ι I ι ι ι ι I ι ι ι ι I

Figure 2. Fractionation of lignin sulfonates in spent sulfite liquor on Sephadex G-75. Eluent: water. (Reprinted with permission from réf. 1. Copyright 1969 Wiley.)

1.0

1


LIGNIN: PROPERTIES AND MATERIALS

126

MOLECULAR WEIGHT (LOG. SCALE)

• SEDIMENTATION VELOCITY INST. OF PAPER CHEMISTRY (USA) • LIGHT - SCATTERING FINN. PULP PAPER RES. INST.


5 0 0 0 0 [30000 20000 10000 5000 3000J-

_L 1.0 2.0 V = EFFLUENT VOLUME

V/Vo 3.0 V = VOID VOLUME 0

Figure 3. M o l a r mass as a function of retention volume for lignin sulfonates. C o l u m n : Sephadex G-50. Eluent: water. (Reprinted with permission from réf. 1. Copyright 1969 Wiley.)

MOLECULAR WEIGHT (LOG. SCALE) 100000 r -

• SEDIMENTATION VELOCITY INST. OF PAPER CHEMISTRY (USA) • LIGHT - SCATTERING FINN. PULP PAPER RES. INST.

50000 30000 20000 10000

500Q£ 1.0 2.0 V = EFFLUENT VOLUME

_L V/Vo 3.0 V = VOID VOLUME 0

Figure 4. M o l a r mass as a function of retention volume for lignin sulfonates. C o l u m n : Sephadex G-75. Eluent: water. (Reprinted with permission from réf. 1. Copyright 1969 Wiley.)


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the m o l a r masses of the l i g n i n sulfonates show a s t r a i g h t l i n e r e l a t i o n s h i p w i t h the r e t e n t i o n volumes (1). In these e x p e r i m e n t s Sephadex G - 5 0 effected f r a c t i o n a t i o n of the l i g n i n sulfonates w i t h m o l a r masses of 5 0 0 0 - 5 0 000 d a l t o n a n d Sephadex G - 7 5 those w i t h m o l a r masses o f 20 0 0 0 - 1 0 0 000 d a l t o n . T h e m o l a r mass det e r m i n a t i o n s c a r r i e d out u s i n g the l i g h t - s c a t t e r i n g a n d u l t r a c e n t r i f u g a l sedi m e n t a t i o n v e l o c i t y methods gave i d e n t i c a l results. H o w e v e r , c a l i b r a t i n g the c o l u m n s u s i n g l i g h t - s c a t t e r i n g or u l t r a c e n t r i f u g a t i o n is too a w k w a r d for r o u t i n e d e t e r m i n a t i o n s . T h e r e is t h u s a need for r e a d i l y available c a l i b r a t i o n s t a n d a r d s o f k n o w n m o l a r mass t h a t elute at the same r e t e n t i o n v o l u m e as l i g n i n sulfonates of the same m o l a r mass. It s h o u l d be noted t h a t the relationships between m o l a r mass a n d ret e n t i o n v o l u m e for l i g n i n sulfonates s h o w n i n F i g u r e s 3 a n d 4 are s t r i c t l y o n l y v a l i d for the samples s t u d i e d i n these e x p e r i m e n t s because l i g n i n s u l fonates are polyelectrolytes a n d t h u s interact w i t h each other a n d w i t h the gel m a t r i x of the c o l u m n . T h e shape of the c a l i b r a t i o n curve is t h u s affected by, a m o n g other t h i n g s , the size a n d c o n c e n t r a t i o n of the s a m p l e (2). Interactions between m o l e c u l a r species c a n be e l i m i n a t e d b y e l u t i n g w i t h a s u i t a b l e electrolyte.

Elution of Lignin Sulfonates with Electrolyte Solution and Calibration of Columns T h e effects caused by the electrolyte n a t u r e of l i g n i n sulfonates are e l i m i n a t e d b y u s i n g a 0 . 5 M s o d i u m chloride s o l u t i o n as eluent. T h i s eluent is m a d e 0 . 1 M w i t h respect to T r i s - H C l a n d buffered to p H 8 w i t h h y d r o c h l o r i c a c i d i n order to dissolve the proteins used as c a l i b r a t i o n s t a n d a r d s ( F i g . 5). T h e c o l u m n is c a l i b r a t e d u s i n g proteins of k n o w n m o l a r mass. T h e relative r e t e n t i o n volumes 0.0 a n d 1.0 are defined b y the e l u t i o n of B l u e D e x t r a n (molecular weight 2 000 000) a n d sulfosalicylic a c i d ( m o l e c u l a r weight 218), respectively. In the e x p e r i m e n t described i n F i g u r e 6, four l i g n i n sulfonate fractions w i t h k n o w n m o l a r masses were eluted. F i g u r e 6 shows t h a t the proteins used for c a l i b r a t i o n elute i n the same way as l i g n i n sulfonates, w h i c h justifies the use of proteins as c a l i b r a t i o n s t a n d a r d s . A c o m p a r i s o n between F i g u r e s 4 a n d 6 shows t h a t e l u t i o n w i t h an electrolyte s o l u t i o n fractionates l i g n i n sulfonates i n the range 3 0 0 0 80 000 d a l t o n , b u t t h a t e l u t i o n w i t h water fractionates those i n the range 20 0 0 0 - 1 0 0 000 d a l t o n . Because of the very h i g h m o l a r masses of e n z y m i c a l l y p o l y m e r i z e d l i g n i n sulfonates, S e p h a c r y l S-300 is used as the gel m a t r i x . T h e f r a c t i o n a t i o n range is 10 000 to 1 000 000 d a l t o n . E v e n i n t h i s case proteins of k n o w n m o l a r mass can be used as c a l i b r a t i o n s t a n d a r d s ( F i g s . 7 a n d 8). O n e disadvantage of u s i n g salt s o l u t i o n as eluent is t h a t the l i g n i n sulfonates tend to adsorb o n t o the gel m a t r i x , r e s u l t i n g i n a r e s o l u t i o n inferior to t h a t o b t a i n e d b y e l u t i o n w i t h water. O n the other h a n d , e l u t i o n b e h a v i o r w i t h water is adversely affected b y the p o l y e l e c t r o l y t e properties o f the l i g n i n sulfonates. A d s o r p t i o n , w h i c h is caused b y the phenolic h y d r o x y l

128


A 80nm I

MOLAR MASS DISTRIBUTION, %


2

0.00

0.20 80000

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1

1

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20000 '

0.80 1.00 1.20 RELATIVE RETENTION VOLUME 10000 5000 — MOLAR MASS 1

1

F i g u r e 5. F r a c t i o n a t i o n o f l i g n i n sulfonates o n e l u t i o n t h r o u g h S e p h a d e x G - 7 5 . E l u e n t : 0 . 5 M N a C l , 0 . 1 M I r i s - H C l ( p H 8).

COLUMN: SEPHA0EX G-75 E L U E N T : TRIS/HCL BUFFER SOLUTION (pH 8.0. 0.1 M) NaCl (0.5 M) Log. M ALBUMIN

5.0i—

FROM HEN EGG M = 45000 C H Y M O T R Y P S I N O G E N A Ms25000

LSA Μ χ 46000 - t LSA

4.0h

^ ^ ^ i ^

f — C Y T O C H R O M C M «12500

M s 25 200 ^

•

•DETERMINED

BY

G L U C A G O N M.3483

LIGHT-SCATTERING

3.0h 0

I

I

I

L

0.20

0.40

0.60

0.80

1.00 REL. RET. VOLUME

F i g u r e 6. C a l i b r a t i o n o f Sephadex G - 7 5 . E l u e n t : 0 . 5 M N a C l , 0 . 1 M T r i s - H C l , p H 8 (4).


9.

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RELATIVE RETENTION VOLUME

^ T - t ^

1 0

*

4.10'

2-W W

W

F i g u r e 7. F r a c t i o n a t i o n o f e n z y m i c a l l y p o l y m e r i z e d l i g n i n sulfonates t h r o u g h S e p h a c r y l S-300. E l u e n t : 0 . 5 M N a C l , 0 . 1 M T r i s - H C l ( p H 8).

LOGM

1 0

I

0.20

I

0.40

I

0.60

I

0.80

I

1.00

I

RELATIVE RETENTION VOLUME (RRV)

F i g u r e 8. C a l i b r a t i o n of S e p h a c r y l S-300. E l u e n t : 0 . 5 M N a C l , 0 . 1 M T r i s - H C l ( p H 8).


130


groups of the l i g n i n sulfonates, can be avoided b y u s i n g a s t r o n g l y alkaline eluent to ionize the phenolic h y d r o x y l groups. However, the Sephadex G - 7 5 used for the f r a c t i o n a t i o n of l i g n i n sulfonates i n the m o l a r mass range 3 0 0 0 80 000 d a l t o n is r a p i d l y degraded b y s t r o n g alkaline s o l u t i o n s . S e p h a c r y l is stable enough, b u t the h i g h m o l a r mass c a l i b r a t i o n proteins cannot be used i n s t r o n g alkaline s o l u t i o n .

Elution of Non-Sulfonated Lignins with Sodium Hydroxide Solution as Eluent N o n - s u l f o n a t e d l i g n i n s such as those f r o m alkaline p u l p i n g processes are insoluble i n water b u t easily soluble i n s o d i u m h y d r o x i d e s o l u t i o n s . W h e n dissolved i n a n d eluted w i t h a s o d i u m h y d r o x i d e s o l u t i o n , they show p o l y electrolyte properties, i.e., the m o l e c u l a r species i n t e r a c t . A s revealed b y F i g u r e 9, the f r a c t i o n a t i o n result is s t r o n g l y dependent o n the s o d i u m h y droxide c o n c e n t r a t i o n u p to a c o n c e n t r a t i o n of 0 . 4 M . A 0 . 5 M s o d i u m h y droxide s o l u t i o n is thus a n a p p r o p r i a t e eluent for f r a c t i o n a t i o n o n Sephadex G - 5 0 (3). W i t h 0 . 5 M s o d i u m h y d r o x i d e as eluent, Sephadex G - 5 0 effects fract i o n a t i o n i n the m o l a r mass range 1000-15000 d a l t o n a n d can be used for a p e r i o d of 3-4 weeks w i t h a single c a l i b r a t i o n carried out w i t h proteins a n d p o l y p e p t i d e s of k n o w n m o l a r mass, as revealed b y F i g u r e 10. R e l a t i v e retention volumes 0.0 a n d 1.0 are defined w i t h B l u e D e x t r a n a n d p h e n o l , respectively. D u e to the dark color of a l k a l i l i g n i n s , their m o l a r masses cannot be d e t e r m i n e d b y means of the l i g h t - s c a t t e r i n g m e t h o d . However, as s h o w n b y F i g u r e 10, e l u t i o n w i t h s o d i u m h y d r o x i d e also brings about a consistent e l u t i o n p a t t e r n of l i g n i n sulfonates a n d p o l y p e p t i d e s . It is assumed t h a t this also applies to the kraft l i g n i n s . F r a c t i o n a t i o n o n Sephadex G - 2 5 using 0 . 5 M s o d i u m h y d r o x i d e as e l u ent causes the low m o l a r mass l i g n i n components i n black l i q u o r to elute i n the r e l a t i v e r e t e n t i o n volume range 0.3-1.3 w i t h p a r t i a l s e p a r a t i o n f r o m each other, as s h o w n i n F i g u r e 11. It s h o u l d be noted t h a t i n t h i s relative r e t e n t i o n v o l u m e range e l u t i o n does not necessarily take place i n order of decreasing m o l e c u l a r size, because, as seen f r o m F i g u r e 12, f u n c t i o n a l groups m a y have a greater effect on e l u t i o n behavior t h a n m o l e c u l a r size (Forss, K . ; T a l k a , E . , T h e F i n n i s h P u l p a n d P a p e r Research I n s t i t u t e , u n p u b l i s h e d results).

Summary It has been s h o w n t h a t the m o l a r mass d i s t r i b u t i o n s of l i g n i n sulfonates a n d kraft l i g n i n can be d e t e r m i n e d by gel p e r m e a t i o n c h r o m a t o g r a p h y . C a l i b r a t i o n of the c o l u m n s w i t h l i g n i n sulfonates of k n o w n m o l a r mass or, a l t e r n a t i v e l y , w i t h c o m m e r c i a l l y available proteins a n d p o l y p e p t i d e s has been s h o w n to give the same r e s u l t . Because of the polyelectrolyte properties of lignins, e l u t i o n is p e r f o r m e d w i t h electrolyte solutions. If the l i g n i n s are water soluble a n d the c o l u m n

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280nm I

ELUENT

RELATIVE RETENTION VOLUME Figure 9. Influence of sodium hydroxide concentrations in the eluent on fractionation of lignins in draft black liquor. Column: Sephadex G-50. (Reprinted with permission from ref. 3. Copyright 1976 Wiley.)

COLUMN: SEPHAOEX ELUENT : 0.5 Μ ΝαΟΗ

f— CYTOCHROM C Ms12500

Log. M

6-50

r— L S A M m 8900 LSA M = 5200

4.00

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GLUCAGON M.3483 BACITRACIN M » U23

3.00

h LSA = LIGNOSULFONATE

Ι

I

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0.40

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F i g u r e 10. C a l i b r a t i o n of Sephadex G - 5 0 . E l u e n t : 0 . 5 M N a O H (4).


132

M

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0.8

F i g u r e 11. F r a c t i o n a t i o n of low m o l a r mass l i g n i n c o m p o n e n t s i n kraft b l a c k l i q u o r . C o l u m n : Sephadex G - 2 5 . E l u e n t : 0 . 5 M N a O H .

ABS0RBANCE

Figure 12. Fractionation of low molar mass model compounds. Column: Sephadex G-25. Eluent: 0.5M N a O H .

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gel m a t r i x is n o t stable enough i n the presence o f a n a l k a l i n e eluent, t h e e l u t i o n is performed w i t h 0 . 5 M s o d i u m chloride s o l u t i o n adjusted t o p H 8 w i t h 0 . 1 M T r i s - H C l buffer. If the lignins are soluble o n l y i n a l k a l i n e s o l u t i o n the e l u t i o n is performed w i t h 0 . 5 M s o d i u m h y d r o x i d e s o l u t i o n . Literature C i t e d 1. Forss, K.; Stenlund, B. Pap. Puu 1969, 51, 93-105. 2. Stenlund, B. Pap. Puu 1970, 52, 55-62; 121-30; 197-206; 333-39; 671-79. 3. Forss, K.; Stenlund, B.; Sågfors, P.-E. Appl. Polym. Symp. 1976, 28, 1185-94. 4. Forss, K . G . ; Furmann, A . G . M . U.S. Patent 4 105 606, 1978. RECEIVED April 3,1989

Determination of the Molar Mass Distribution of ... - ACS Publications

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